The HIV-1 proteome consists of required structural and essential regulatory proteins as well as accessory proteins that increase viral fitness. These accessory proteins, Vif, Vpr, Vpu, and Nef, are involved in countering host immune responses and antagonizing so-called restriction factors, thereby increasing viral infectivity, the ability of a virion to infect and replicate in target cells. Nef, or negative factor, has a variety of activities, most of which involve modulation of signal transduction or membrane protein sorting. Nef increases the infectivity of viral particles (virions), with a mechanism that depends on cellular endosomal proteins as well as on sequences within the viral Envelope glycoprotein (Env). Two members of the serine incorporator (SERINC) protein family were identified recently as host proteins that inhibit viral infectivity and are antagonized by Nef. SERINC5 (SERC5) is the most active; it incorporates into virions and inhibits virion infectivity, potentially by altering the kinetics of virion-target cell fusion, the function of the Env protein. Despite the prevailing model that SERC5 incorporates into virions to inhibit Env function, it does not seem to co-immunoprecipitate with Env, suggesting the requirement of cofactors or other indirect interactions. Moreover, different Env proteins are differentially sensitive to SERC5, but the structural and molecular determinants of SERC5-sensitivity are unclear. To inhibit SERC5 antiviral activity, Nef coordinates with endosomal machinery to relocalize SERC5 from the plasma membrane to late endosomes. However, this model of SERC5-inhibition is derived solely from experiments in which exogenous SERC5 is expressed. Whether endogenous SERC5 is similarly modulated is unknown. Here studies are proposed to determine the mechanisms behind SERC5-mediated restriction of virion infectivity and Nef-mediated counteraction of this effect. Preliminary analysis of clade B Envs leads to the hypothesis that a region within the gp41 membrane proximal external region (MPER) is responsible for SERC5-sensitivity. This region and other putative determinants will be probed by site-directed mutagenesis. To determine whether a direct interaction of SERC5 with Env can explain antiviral activity, or instead whether SERC5-cofactors are responsible, a proximity-based labeling approach using APEX2, coupled with mass spectrometry-based proteomics, will be used to probe the interactions of SERC5 and Env. Lastly, the hypothesis that Nef downregulates endogenous SERC5 from the cell surface and traps it in late endosomes via clathrin-mediated endocytosis will be tested using cell fractionation by differential centrifugation and targeted proteomics to track the native, endogenous, unlabeled protein. When these experiments are completed, the field will have a better explanation of the effect of SERC5 on HIV-1 virion infectivity. This understanding might provide new approaches to restricting transmission, a pillar of the NIAID mission.